Most normal human cells undergo a limited number of cell divisions, eventually entering an irreversibly arrested state, through either senescence or differentiation. Both processes have major implications for human health; between them impacting birth defects, cancer and the degenerative effects of human aging. For example, defects in cell differentiation during embryo development result in human birth defects. Senescence and differentiation programs are both characterized by profound changes in chromatin structure, and, in both cases, this is thought to contribute to the altered cell phenotype. We are using senescence as a model system to study these changes in chromatin structure and their contribution to two hallmarks of both senescence and terminal differentiation, repression of proliferation-promoting genes and cell cycle exit. Recently, we showed that the chromatin regulatory protein, HIRA, and its physical binding partner, ASF1a, both play a key role in formation of a novel chromatin structure in senescent cells, called senescence associated heterochromatin foci (SAHF). SAHF is thought to silence genes that drive cell proliferation. HIRA and ASF1a drive SAHF formation, acting in concert with a subnuclear organelle, the PML body; and two chromatin associated proteins, HP1 and macroH2A. Preliminary data indicate that the HIRA/ASF1a pathway is activated by the key proliferation-regulating kinase, GSK3. To understand the physiological significance and molecular basis of SAHF formation and its mode of activation in presenescent cells, we will use cell and molecular biology techniques to: Specific Aim 1. Investigate the structure of SAHF, its mechanism of assembly by HIRA/ASF1a and PML nuclear bodies and identify its key growth suppressor components. Specific Aim 2. Investigate the function and mechanism of incorporation into SAHF of chromatin associated proteins, HP1 and macroH2A. Specific Aim 3. Investigate the role of GSK3 activity in localization of HIRA to PML bodies, formation of SAHF and onset of senescence. [unreadable] [unreadable]